The aim of this study was to evaluate the removal of the smear layer by some commonly used (needle-and-syringe irrigation, sonic activation, ultrasonically activated irrigation) and new root canal irrigation strategies (negative pressure irrigation and polymer rotary file) using a novel approach by comparing pre- and post-experimental images. Prepared root canals (n = 50) were subjected to a split tooth model and divided into 5 groups (n = 10): (1) needle-and-syringe irrigation (control); (2) sonic activation (SA); (3) negative pressure irrigation with continuous warm activated irrigation and evacuation (CWA); (4) polymer finishing file (FF); (5) ultrasonically activated irrigation (UAI). Smear layer scores and percentage of open dentinal tubules (%ODT) were evaluated by 2 examiners before and after irrigation procedures, from the middle and apical thirds of the root canal, on scanning electron microscopic images. Data were analysed using Kruskal-Wallis and post hoc tests at P = 0.05. Needle-and-syringe irrigation (control) showed no significant difference (both smear score and %ODT) compared to the pre-experimental value (P > 0.05). All other groups showed lower smear scores and higher %ODT, compared to the control (P < 0.05). The lowest smear score and highest %ODT were observed in the CWA group, which was significantly different from all other groups (P < 0.05). SA group showed significantly higher smear scores and lower %ODT than FF or UAI (P < 0.05). CWA showed superior removal of smear layer in the middle and apical thirds of the root canal compared to the other irrigation strategies.